Project Summary/Abstract Most adult mammalian tissues and organs have very limited regenerative potential. In patients with a heart attack, the death and loss of heart muscle cells is irreversible and often results in permanent scarring and potentially life-threatening arrhythmias. In contrast, neonatal mice and adult zebrafish are able to rapidly regenerate their hearts. Genetic lineage-tracing experiments have revealed proliferation of pre-existing cardiomyocytes as the dominant mechanism to generate new muscle cells. However shortly after birth, the majority of cardiomyocytes in most mammalian species undergoes a last round of DNA replication without cytokinesis, become binucleated, and withdraw from the cell cycle. What physiological signals trigger mammalian cardiomyocyte perinatal binucleation and cell cycle arrest, and how these stimuli are differentially regulated in animals with distinct cardiac regenerative potentials are among the most long-standing questions in cardiomyocyte biology. Our preliminary observations from comparative analyses of cardiomyocytes across phylogeny, in vivo chemical screens of candidate pathways, together with functional studies in both mice and zebrafish suggest a critical role of the perinatal changes of endocrine systems in driving cardiomyocyte proliferative and regenerative potential loss in the mammalian heart. In this proposal, we plan to combine a novel cardiomyocyte quantification assay with state-of-art genetic tools to investigate the functions of nuclear hormone receptor activation in regulating cardiomyocyte proliferation during postnatal growth (Aim 1) and heart regeneration following myocardial injury (Aim 2). In addition, we will examine the underpinning cellular and molecular basis, and determine the function of novel downstream target genes in cardiomyocyte cell cycle control through gain- and loss-of-function approaches (Aim 3). Successful completion of the proposed work will thus reveal mechanisms underlying the loss of cardiomyocyte regenerative potential in ontogeny and phylogeny.